Project Summary/Abstract Central to the function of macromolecules are the conformational dynamics they undergo in order to carry out biological function. Native mass spectrometry (MS), whereby non-covalent interactions are preserved in the mass spectrometer, is emerging as a powerful technique to study protein stoichiometry, topology, dynamics, and kinetics and protein-ligand interactions. Native MS coupled with ion mobility (IM-MS), which reports on macromolecule shape, is revolutionizing how large conformations of proteins and protein complexes are analyzed and understood. However, existing commercial IM-MS instrumentation is limited by relatively low resolving powers that render their ability to delineate different structures based on differences in their shapes. This proposal describes a program for developing a small, multipass selected overtone mobility spectrometry (M-SOMS) device that can be inserted into commercial platforms commonly used for structural studies. The aim is to improve resolving power in the first year by a factor of 2 to 5 fold; ultimately the resolving power of the M-SOMS device will be tunable, such that high-resolution spectra (having resolving powers that are more than an order of magnitude greater than currently available) will be accessible to any researchers using the commercial platforms. The M-SOMS device will be developed, optimized, and validated using the monomeric and oligomeric ubiquitin system (as well as metallothionein?metal complexes) and applied to tackle larger, more complex protein and protein-ligand systems. Specifically, the high-resolving power will make it possible to discern small conformational differences for the oncoprotein RAS in complex with guanidine nucleotides and analogs, as well as other effector proteins. Lastly, the membrane protein aquaporin, a tetrameric water channel, in complex with lipids will be investigated with the M-SOMS instrument. The latter two studies will represent the first high-resolution mobility study of an intact protein complex. We envisage that M-SOMS will have a significant impact in structural biology and related fields by enabling a number for conformational states to be captured and providing high-resolution mobility restraints for molecular modeling.